L. plantarum strain was isolated from Tunisian traditional fermented milk (ricotta cheese), identified with a conventional method using Api-50 CHL system (BioMerieux, Marcy-l’Étoile, France) and by polymerase chain reaction (PCR) technique using L. plantarum-specific primers: forward IDL04F: 5′-AGGGTGAAGTCGTAACAAGTAGCC-3′ and reverse IDL62R 5′-CTAGTGGTAACAGTTGATTAAAACTGC-3′, giving a product size of about 428bp as described previously.19

Cell-free supernatant was obtained by centrifuging (10,000×g for 10min at 4°C) L. plantarum culture, grown in MRS broth for 16h at 30° C. The supernatant was adjusted at neutral pH and filter-sterilized (0.22μm, Millipore). The obtained L. plantarum extract (LPE) was conserved at 4°C until use.

Antibacterial activity of LPE was tested against nine strains (Table 1) isolated from the oral cavity of Tunisian children and S. aureus ATCC 25923 using the broth microdilution method.20,21 The LPE was tested alone or in combination with antibiotics.

The S. aureus strains (n=9) used in this study were isolated from the oral cavity of Tunisian children from the dental clinic of dentistry (Monastir, Center of Tunisia).

The criteria for inclusion were: no antibiotic treatment four weeks prior to sampling, no use of mouth rinses or any other preventive measure that might involve exposure to antimicrobial agents, and no systemic disease. A sterile swab was used for sample collection from the oral cavity of each patient. After incubation (24h at 37°C), swabs were plated on blood agar plates supplemented with 5% sheep blood (24h at 37°C) and bacterial identification was achieved using conventional methods.

The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of LPE (5–90%, v/v), BC (Acros organics, USA) (0–1024μg/mL), CHX (Sigma–Aldrich, USA) (0–1024μg/mL) and tetracycline (TET) (0–1024μg/mL) according to the Clinical and Laboratory Standards Institute (2006) guidelines.22

After 24h of incubation, bacterial growth was evaluated by the presence of turbidity and a pellet on the well bottom. MIC was defined as the lowest concentration of the compound that had no macroscopically visible growth. All experiments were carried out three times.

To determine the MBC values, 10μL of each well medium, with no visible growth was removed and inoculated in Muller Hinton agar plates. MBC was defined as the lowest concentration at which 99% of the bacteria were killed. Each experiment was repeated at least twice.23

To determine the potential effect of LPE to modulate drug resistance of S. aureus, MICs of BC, CHX and TET (ranging from 0.5 to 2048μg/mL) were determined alone and combined with a sub-MIC of LPE (1/2 MIC, v/v) using the microtiter plates assay.24

The anti-adhesion properties of LPE (ranging from 10% to 90%, v/v) to S. aureus strains were tested as previously described by Merritt et al.25 Briefly, the bacterial culture was grown in Tryptone soy broth (TSB) at 37°C for 24h. Two microliters were disposed into each well of 96-well plates in the presence of 198μL of the medium supplemented with 2% glucose (w/v) containing LPE. Plates were incubated in aerobic conditions at 37°C for 24h. The anti-biofilm activity of TET, CHX, and BC ranging from 2 to 1024μg/mL were determined against S. aureus strains with or without sub-MIC of the LPE (1/2 MIC, v/v), using the microtiter plates assay. After incubation, the plate was washed with phosphate buffer saline, stained with 100μL of 1% (w/v) crystal violet and incubated at room temperature for 15min. Then, the absorbance at 590nm was determined using a microplate reader (D.E.E.D reader, Bio-Rad instrument). The mean absorbance (OD590nm) of the sample was determined, and the percentage of inhibition obtained for each concentration of LPE and drugs was determined by the following formula:

To evaluate biofilm inhibition potency of LPE, S. aureus ATCC 25923 was grown in Tryptone soy broth (TSB) at 37°C for 24h and then diluted into the TSB medium (105cells/mL) supplemented with 2% glucose (w/v). For each concentration of the tested drugs (2–1024μg/mL) and LPE (5–90%), 10mL of bacterial suspension was incubated in a 6-well plate containing round glass cover slips for 24h at 37°C. After adhesion, the non-adhering bacteria were removed by rinsing the substrate four times with sterile PBS. Then the substrate was air dried and observed using AFM. All AFM images were then processed to analyze the biofilm formation behavior.

Data were analyzed using SPSS v. 17.0 software. The Friedman test, followed by the Wilcoxon signed ranks test were used to assess inter-group differences. In addition, statistical significance was set at p-value <0.05.

As presented in Table 1, LPE demonstrated selective antimicrobial properties. Seven S. aureus strains (S3, S4, S5, S6, S1, S8 and S9) and S. aureus ATCC 25923 have a MICs value ranged from 20% to 50% (v/v). The remaining two strains (S2, S7) were more susceptible (MIC 10%). In addition, a variable MBCs (10–90%) (v/v) was observed according to the tested strain (Table 1). On the other hand, out of the nine oral S. aureus strains, eight (Table 1) were considered resistant to BC (MICs between 4 and 128μg/mL) and only one strain (S4) was sensitive (MICs≤2μg/mL); all strains were resistant to CHX (MICs>2μg/mL). Moreover, only one strain (S4) was susceptible to TET (MIC<2μg/mL).

Data presented in Table 1 showed that the presence of LPE at 1/2-MIC (v/v) in combination with BC resulted in a 4-fold reduction of MICs and MBCs of S. aureus ATCC 25923 and one oral strain (S6). Concerning the synergistic effect of CHX and LPE, there was an important diminution of MIC and MBC of CHX (2–8-fold potentiation) in six strains (Table 1). Furthermore, a 2-8-fold reduction of MIC (μg/mL) of TET against seven strains when combined with LPE was noticed (Table 1).

Biofilm formation of oral S. aureus strains was evaluated in 96 microtiter wells plate with LPE (ranging from 5% to 90%, v/v). Results were expressed as minimum biofilm inhibition concentration (BIC50 and BIC90). As presented in Table 2, BIC90 of LPE was reached at 54% LPE supplementation, for two oral strains (S4 and S7). While, BIC50 ranged from 28 to 77% (v/v) LPE supplementation (Table 2).

In addition, BC showed various effects on the development of S. aureus biofilms with BIC50 values ranging from 12 to 112μg/mL for the tested strains. Moreover, the combination of BC and 1/2 MIC LPE resulted in a reduction of BIC50 against eight strains (Table 2). In the same way BIC90 was also reduced with the combination of BC and LPE.

Concerning the effect of CHX alone and in combination with LPE, BIC50 values ranged from 34–355μg/mL for CHX for the tested strains (Table 2) whereas the combination of CHX and 1/2 MIC of LPE % (v/v) exhibited a reduction of BIC50 and BIC90 against all the tested strains (Table 2).

Our results revealed that TET may reduce S. aureus attachment to polystyrene (BIC90 reach 30μg/mL for S4). In addition, the combination of TET and 1/2 MIC of LPE exhibited a reduction of BIC50 and BIC90 against nine strains (Table 2).

AFM revealed that the tested strains were strongly biofilm positive and had the potency to adhere to glass surface (Fig. 1A). On the other hand, LPE decreased significantly the adherence to glass (Fig. 1D). In addition, the combination of LEP with TET, BC and CHX decreased significantly the biofilm formation (Fig. 1B–E). As presented in Fig. 1F, almost total biofilm inhibition was observed with 16μg/mL of BC combined with 1/2 MIC of LPE % (v/v).

Fig. 1.

AFM images of biofilm formed by S. aureus ATCC 25923 to glass surface: (A) TSB alone; (B) TSB with 4μg/mL of BC+1/2 MIC LPE; (C) TSB with 8μg/mL of CHX+1/2 MIC LPE; (D) TSB with+40% LPE (v/v); (E) TSB with 8μg/mL TET+1/2 MIC LPE; (F) TSB with 16μg BC+1/2 MIC LPE.

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